Astrocytes provide physical and metabolic support for neurons in the central nervous system. Reactive astrocytes, however, play a crucial role in neuroinflammation after brain injury. Therapeutic hypothermia has been shown to be neuroprotective in brain injury patients. In order to understand the effect of therapeutic hypothermia on astrocytes in stroke patients, we performed a proteomic profiling of astrocytes following transient focal cerebral ischemia in rats under normothermic (37 degrees C) or mild hypothermic condition (33 degrees C). Primary astrocytes were prepared from rat brain using a Percoll gradient method, and their proteome profiles were determined by using LC/ESI-MS/MS followed by high-throughput label-free quantification. Comparison of proteome profiles of astrocytes following transient focal ischemia revealed that hypothermia upregulated (=1.5-fold) 86 proteins and downregulated (=0.6-fold) 47 proteins compared to normothermic condition. The changes of protein expression were validated by Western blot or RT-PCR. Ingenuity Pathways Analysis and Database for Annotation, Visualization and Integrated Discovery analyses of the up- or downregulated proteins indicate that hypothermia influences glutamatergic signaling, cell death, and stress response of astrocytes in the ischemic brain. Therefore, therapeutic hypothermia may be neuroprotective in stroke patients by modulating astrocytic functions and survival.